Mentha arvensis Linn./Japanese Mint Plant.
cv. Saksham.
The present invention is related to the development of a novel high menthol producing plant obtained through a unique method of screening of the somaclones in poison agar medium containing toxic level of menthol. The selected plant is tolerant to high concentration of menthol in cultures and hence possesses property of accumulating more menthol per unit leaf mass due to shifted feed back inhibition by menthol as end product. The essential oil yield from the said plant is high coupled with the property of being rich in menthol. This plant is unique and clearly distinct from all other existing varieties of Mentha arvensis L. The new variety was initially designated GRB 30. The new variety has been named as xe2x80x98Sakshamxe2x80x99 (meaning capable) which can be propagated vegetatively through suckers for commercial cultivation.
Mentha arvensis Linn. Var piperescens. Holmes (menthol or Japanese mint) is a highly valued industrial crop due to menthol, which is purified by crystallisation through freezing from its essential oil. In the varietal improvement programs, the genetic alternations leading to enhancement in the menthol content in the essential oil and improving other adaptive characters determining the yield and quality of essential oil are most desirable. Menthol is produced from geranyl pyrophosphate through a series of intermediates and the level of production of the high value product menthol is controlled through feed back inhibition of the accumulated end product (which is menthol here). It appears that beyond a certain limit of menthol during the biosynthesis the enzymes involved are inhibited by the end product preventing further forward reaction. Also, monoterpenes are known to be cytotoxic to plant tissues, by inhibiting respiration and photosynthesis by drastically affecting the mitochondria, golgi bodies etc and decreasing cell membrane permeability (Brown J T, Hegarty P K, Charlwood B V, 1987. The toxicity of monoterpenes to plant cell cultures. Plant Science 48: 195-201). Monoterpenes are either sequestered in the plants in specialized structures like glandular hairs in Pelargonium (Brown J T, Charlwood B V, 1986, Differentiation and monoterpene biosynthesis in plant cell cultures. In Morris P, Scragg A, Stafford A and Fowler M, ed Secondary Metabolism in Plant Cell Cultures. Cambridge University Press, Cambridge, 1986, p. 68), trichomes in Mentha or stored in the form of non-toxic glycoside derivatives in vacuoles e.g. Rosa spp. The end product toxicity in such cases can be related to the feed back inhibition to regulate the extent of monoterpenes that can be accumulated within tolerance limits. Menthol, the major component of the essential oil, which is considered to be cytotoxic to the plant, could be so due to toxicity mechanism related to the end product. To this corollary, the level of menthol accumulated in leaf tissues may be linked to the level of menthol tolerance. Uncoupling of these two factors may thus deregulate the rate-limiting step in the production of menthol. With this rationale, the applicants attempted to screen the somaclones available with us and tested to select clones tolerant to high level of menthol with the possibility of selecting high menthol yielding clones. It was assumed that through certain alternate mechanism cellular components may escape toxicity and damaging effect of higher menthol in the tissues by circumventing feed back inhibition level. Such a condition may lead to more menthol accumulation in the trichomes of the regenerated menthol tolerant plant which indeed happened and the applicants could screen out a stable high menthol containing clone showing enhanced tolerance to menthol in vitro.
The main object of the invention is to develop a new and distinct plant through tissue culture, said plant being capable of producing higher menthol with high essential oil yield as well as herbage yield.
Another object of the invention is to develop a new plant which possesses better vegetative growth with high regenerability.
Still another object of the invention is to retain certain original characters of the parent plant.
This invention provides a new and distinct mint plant of Mentha arvensis xe2x80x98Sakshamxe2x80x99, developed through tissue culture, possessing the following combination of characters namely producing higher amount of menthol with high essential oil yield as well as herbage yield; possesses better growth and vegetative growth with high regenerability; has distinct molecular profile by random amplified polymorphic DNA (RAPD); retains the characteristics of tolerance to leaf spot, rust and powdery mildew as in the parent variety xe2x80x98Himalayaxe2x80x99; and has light greenish leaves, pinkish white flowers like the parent plant xe2x80x98Himalayaxe2x80x99. This new plant is able to produce higher herbage, oil and menthol yield per unit area as compared to other existing improved varieties.
The new plant xe2x80x98Sakshamxe2x80x99 of the present invention was produced at the Central Institute of Medicinal and Aromatic Plants field station Pant Nagar, Lucknow, India.
The new variety of the present invention commonly produces a significant number of suckers, and has been asexually reproduced at the same location in India by division. The xe2x80x98Sakshamxe2x80x99 plant is firmly fixed in its characteristics and has been found to reproduce true to type through successive generations of such asexual reproduction.
The invention provides a new and distinct mint plant of Mentha arvensis xe2x80x98Sakshamxe2x80x99, developed through tissue culture, possessing the following combination of characters:
a) Produces higher amount of menthol (83% of oil) with high essential oil yield (0.8 to 1.0%) as well as herbage yield (at least 1.83 Q per 100 m2),
b) Possesses better growth and vegetative growth with high regenerability covering at least 77 cm canopy area and a height of at least 72 cm in a maximum of 100 days,
c) Has a distinct molecular profile by random amplified polymorphic DNA (RAPD) using 20 random primers distinguishing the plant from the other existing varieties,
d) Retains characteristics of tolerance to leaf spot, rust and powdery mildew as in the parent variety xe2x80x98Himalayaxe2x80x99.
e) Has light greenish leaves (138B), pinkish white flowers (56C) like the parent plant xe2x80x98Himalayaxe2x80x99, and
f) Produces higher herbage, oil and menthol yield per unit area as compared to other existing improved varieties.
The applicants used the plant xe2x80x98Himalayaxe2x80x99 (U.S. Plant Pat. No. 10,935), an elite mint genotype for large scale screening of in vitro raised clones (Khanuja S P S, Shasany A K, Dhawan S, Sushil Kumar, 1998, Rapid procedure for isolating somaclones of altered genotypes in Mentha arvensis. J medicinal and Aromatic Plant Sciences 20:359-361) to select clones tolerant to high menthol concentration in the medium (An efficient in vitro selection procedure for large scale screening of mint clones to isolate high menthol genotypes. Khanuja et al, Procedure patent pending, 1999).
While testing the tolerance of in vitro regenerates in presence of menthol in the MS based medium (Murashige T and Skoog F, 1962, A revised medium for rapid growth and bioassay with tobacco tissue cultures; Physiol. Planta. 15 473-497), the initial toxicity symptoms were first observed at the menthol concentration of 40 xcexcg mlxe2x88x921 menthol after 7 days. The immediate symptoms of menthol toxicity were observed at menthol concentration of 70 xcexcg mlxe2x88x921 within 24 hours. Here it was immediate chlorophyll loss and irreversible wilting of the shoots, non-rescuable even by culturing these regenerates in normal medium without menthol. Lowest survival was detected at a concentration of 70 xcexcg mlxe2x88x921, where only 1% shoots could survive. Since at 50 xcexcg mlxe2x88x921 concentration of menthol, also typical symptoms of toxicity were observed, a stepwise selection pressure of menthol concentration in the medium was employed in the large scale screening experiment, to enrich for the potentially tolerant shoots which presumably resort to the adaptive response by tolerating this medium level of menthol concentration through the induction of genes required for the tolerance, before transfer to the higher level. Ultimately 36, clones were selected for tolerance at 70 xcexcg mlxe2x88x921 menthol by screening about 2950 clones. These were transferred to field for evaluation in a plant test area at Lucknow, India. Out of all the clones selected and grown outdoors in the field, one clone GRB-30 was observed to be growing better with high essential oil yield and higher menthol content. Further, on retesting in vitro, this clone could tolerate even 80 xcexcg mlxe2x88x921 menthol in the culture medium. Considering the stability of the multiplied clone for menthol tolerance and increased menthol production it was logically assumed that the mother cells in vitro might have undergone some genetic changes to give rise to these variant (clones) and as a consequence of selection pressure of menthol, ultimately got selected as menthol tolerant genotypes with much higher tolerance level than the control plant xe2x80x98Himalayaxe2x80x99. The selected clone named xe2x80x98Sakshaxe2x80x99 (meaning capable) demonstrated significantly high menthol production per unit biomass and hence represents the superior genotype with the capability for large-scale commercial cultivation. The comparative field evaluation for two years 1998-1999 and 1999-2000 for growth and yield traits is given in Table 1, where the checks included parent variety xe2x80x98Himalayaxe2x80x99 and another superior genotype xe2x80x98Kosixe2x80x99 (non-patented in the United States) in addition to a high menthol tolerant but low herbage yielding variety xe2x80x98Kalkaxe2x80x99 (non-patented in the United States).